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Larsen, R. C., Kaiser, W. J., Wyatt, S. D., Buxton-Druffel, K. L., and Berger, P. H. 2003. Characterization of a new potyvirus naturally infecting chickpea. Plant Dis. 87:1366-1371. During the 1999 to 2001 growing seasons, symptoms consisting of mosaic, stunting, yellowing, wilting, shortening of internodes, and phloem discoloration were observed in chickpea (Cicer arietinum) grown in the Department of Chuquisaca in southern Bolivia. In some fields, approximately 10% of the plants exhibited viruslike symptoms and suffered greatly reduced seed yields. Lentil (Lens culinaris) was also observed to be infected but not pea (Pisum sativum) or faba bean (Vicia faba) growing in nearby fields. Infected chickpea tissue reacted positively to the potyvirus group-specific monoclonal antibody (MAb), but there was no serological reaction with antisera to the potyviruses Bean yellow mosaic virus, Clover yellow vein virus, Cowpea aphid-borne mosaic virus, Pea seedborne mosaic virus, Bean common mosaic virus, or Bean common mosaic necrosis virus. Western blots of total protein extracts probed with the potyvirus MAb revealed a single band ca. 32 kDa. Comparative sequence analysis of cDNA clones generated from the putative coat protein gene consisted of 282 amino acids (31.9 kDa) and showed moderate identities of 67, 66, 63, 63, and 61% with the coat proteins of potyviruses Pepper severe mosaic virus, Pepper yellow mosaic virus, Potato virus Y, Plum pox virus, and Pepper mottle virus, respectively. Phylogenetic analysis of the coat protein amino acid sequence revealed that this virus is a unique member of the family Potyviridae and is phylogenetically most closely related to a group of Solanaceae-infecting potyviruses rather than to other legumeinfecting potyviruses. The proposed name for the new causal agent is Chickpea yellow mosaic virus.

Bean common mosaic virus (BCMV) and bean common mosaic necrosis virus (BCMNV) belong to the genus Potyvirus and are probably the most economically important viruses affecting common bean (Phaseolus vulgaris L.). Of the two viruses, BCMNV has been the most important in common bean production in North America, East Africa and Europe due to significant yield losses incurred from infected fields [10]. BCMV and BCMNV are transmitted in a non-persistent manner by several aphid species. All known strains of each virus can be highly seed-transmitted in bean and are distributed worldwide, in large part as a result of movement of contaminated seed [4, 5]. Drijfhout [4] originally separated the two viruses and their associated strains into eight different pathogroups based on host reaction to infection under specific temperature regimes. Strains NL-5 and NL-3 from Africa were originally assigned to the BCMV pathogroup VI by Drijfhout [4], and NL-8 was assigned to pathogroup III according to host responses in 12 different common bean host groups. The TN-1 strain from Tanzania was later assigned to pathogroup VI by Silbernagel [12]. Prior to 1992, all strains of BCMNV were designated as BCMV, at which time Vetten et al. [14] designated stains NL-3, NL-5 and NL-8 as serogroup A (BCMNV). All other known strains were assigned to serogroup B (BCMV) based on serological reactions to two highly specific monoclonal antibodies, and this was further supported by molecular analysis and ultrastructural comparisons [14]. Currently, several isolates of strain NL-3 have been sequenced and are available in GenBank; however, the sequences of strains NL-5, NL-8 and TN-1 are unavailable. Therefore, we report here the complete genome sequences of these three strains of BCMNV. Common bean seeds (variety ‘Dubbele Witte’) infected with NL-5, NL-8 and TN-1, respectively, were germinated in pots containing a mixture of peat moss and perlite (Sun Gro Horticulture Canada Ltd.) in the greenhouse, and plants were observed for symptoms. Tissues from symptomatic seedlings were evaluated for infection with BCMNV by direct ELISA using monoclonal antibodies specific for serogroup A, and also to serogroup B [11] to verify that there was no contamination with any strain of BCMV. Total nucleic acids were extracted from plant tissues, and cDNA was produced by reverse transcription using MMLV reverse transcriptase (Promega Corp., Madison, WI). Thermocycling parameters were optimized, and a final profile was employed that consisted of a single cycle of 2 min at 94 C; 25 cycles of 30 s at 94 C, 30 s at 50 C, and 3 min at 72 C; and a final extension for 7 min at 72 C. The initial reactions included oligo-dT24 as the reverse primer and a degenerate forward primer, 5’-GAA YAG CAA TGC NAT AG-3’, that produced a fragment approximately 3425 bp in length. The degenerate primer begins at nucleotide 6206, located in the NIa protein region. Subsequent reactions to complete the genome sequence were carried out using custom primer combinations based on the partial nucleotide sequence data obtained. The 5’-proximal region of the genomic sequence R. C. Larsen (&) USDA-ARS, 24106 N. Bunn Rd., Prosser, WA 99350, USA e-mail: richard.larsen@ars.usda.gov

The genome structure and organization of a new and distinct caulimovirus that is widespread in dahlia (Dahlia variabilis) was determined. The double-stranded DNA genome was ca. 7.0 kb in size and shared many of the features of the members of the genus Caulimovirus, such as the presence of genes potentially coding for the movement protein, the inclusion body protein, and the reverse transcriptase (RT), and an intergenic region consisting of a potential 35S promoter. However, the virus differed from the previously described dahlia mosaic caulimovirus and other known caulimoviruses in that the aphid transmission factor (ATF) was absent and the putative coat protein contained a C-terminal deletion and was fused in-frame with the RT. Sequence identity at the amino acid level with known caulimoviruses including a previously reported caulimovirus from dahlia was low and ranged from 32 to 72%. The absence of an ATF and the highly divergent nature of the genomic sequence are characteristics of this new caulimovirus that is widely associated with dahlia.

Polymerase chain reaction (PCR) was applied to detect and establish provisional identity of begomoviruses through amplification of a approximately 575 bp fragment of the begomoviral coat protein gene (CP), referred to as the 'core' region of the CP gene (core CP). The core CP fragment contains conserved and unique regions, and was hypothesized to constitute a sequence useful for begomovirus classification. Virus relationships were predicted by distance and parsimony analyses using the A component (bipartite viruses) or full genome (monopartite viruses), CP gene, core CP, or the 200 5'-nucleotides (nt) of the CP. Reconstructed trees and sequence divergence estimates yielded very similar conclusions for all sequence sets, while the CP 5'-200 nt was the best strain discriminator. Alignment of the core CP region for 52 field isolates with reference begomovirus sequences permitted provisional virus identification based on tree position and extent of sequence divergence. Geographic origin of field isolates was predictable based on phylogenetic separation of field isolates examined here. A 'closest match' or genus-level identification could be obtained for previously undescribed begomoviruses using the BLAST program to search a reference core CP database located at our website and/or in GenBank. Here, we describe an informative molecular marker that permits provisional begomovirus identification and classification using a begomoviral sequence that is smaller than the presently accepted, but less accessible CP sequence.

The 3'-terminal nucleotide sequences of thirteen authenticated strains of bean common mosaic virus (BCMV) and one strain of bean common mosaic necrosis virus (BCMNV) were obtained. The regions sequenced included the coat protein coding sequence and 3'-end non-coding region. These data, combined with sequence information from other legume-infecting potyviruses and the Potyviridae were used for phylogenetic analysis. Evidence is provided for delineation of BCMNV as distinct from BCMV and the inclusion of azuki mosaic, dendrobium mosaic, blackeye cowpea mosaic, and peanut stripe viruses as strains of BCMV. This relationship defines the members of the BCMV and BCMNV subgroups. These data also provide a basis upon which to define virus strains, in combination with biological data. Other aspects and implications of legume-infecting potyvirus phylogenetics are discussed.

Twenty-seven of 29 strains of viruses in the bean common mosaic virus (BCMV) subgroup of legume-infecting potyviruses reacted strongly with one or more of the monoclonal antibodies (MAbs) which are known to be specific for epitopes located along the 50 amino acids which constitute the N-terminal end of the viral coat protein. Approximately one half of the virus strains reacted with the N-terminal epitope specific (NTES) MAb 4G12 which is specific for epitope E/B4, while the other half reacted with NTES MAbs 4 Aff1 or 4F9 which are specific for epitope E/B3. All but two strains contained at least one of these epitopes while no strain contained both. Competitive assays using five sequential, non-overlapping, synthetic, 10mer peptides indicated that the amino acids critical for epitope E/B3 reaction were located at positions 5, 7, and 10 from the N-terminal end of the coat protein. By deduction we postulate that the amino acids critical for epitope E/B4 are located at positions 10, 16, and 17. Because epitope E/B3 requires isoleucine at position 10 for expression whereas epitope E/B4 requires valine to be expressed, no one strain can express both epitopes. Two viruses in our tests (azuki mosaic and Dendrobium mosaic viruses) had deletions in this portion of their sequence explaining their failure to react MAbs specific for either epitope. The critical amino acids for a third epitope, E/B3A, were located at positions 16 and 17. We found no correlation between any of the three N-terminal epitopes defined in this study and the presence or absence of any biological property that we could accurately measure: i.e., symptomatology, host range, or pathotype. However, when coat protein sequences were aligned according to epitope type E/B3 or E/B4, we found that sequences within groups had high levels of identity while between group identities were low. We also found that sequences in the 3′-end non-coding region exhibited similar relationships within and between epitope groups. Two strains of BCMV (NL-4 and RU-1) were found to possess coat protein sequences typical of epitope E/B4 but 3′-NCR sequences typical of epitope E/B3. These data suggest that both strains may be the result of natural recombinants between the two epitope groups.

DNA copies of a wide range of RNA viruses can be made by the direct addition of appropriately treated, purified virus particles to a reverse transcription reaction. Therefore, many problems associated with RNA isolation can be circumvented. Virus particles can be sufficiently destabilized by adjustments of salt content, buffer, pH or by the use of physical force supplied by a freeze/thaw cycle so that RNA in sufficient quantity and physical condition is available for the synthesis of in some cases, full length cDNAs. cDNAs have been made of viruses in the bromo-, poty-, carla-, ilar-, potex-, tobra and tobamovirus groups. Reported here are experiments with cowpea chlorotic mottle virus and bean common mosaic virus.

The DNA of several monopartite and bipartite whitefly-transmitted (WFT) geminiviruses was amplified from a viral template present in infected leaves after either direct addition of clarified plant extracts to an otherwise complete polymerase chain reaction (PCR) mix or after immobilization of template to microfuge tubes. A degenerate primer pair was designed to specifically target the middle or 'core' region of the capsid protein gene of subgroup III geminivirus isolates and amplify a viral DNA fragment of approximately 550 bp. Using this method, a single PCR product of the expected size (550 bp), as estimated by agarose gel electrophoresis, was amplifiable from plants infected with a representative set of subgroup III geminivirus isolates with a broad biogeographic base. That the 550-bp PCR product had a geminiviral gene origin was demonstrated by direct sequencing of the 550-bp fragments (yielding approximately 470 to 490 bases of informative sequence) and was validated through comparison (alignment) of the sequences with the published DNA sequences of several well-characterized WFT geminiviruses. Analogous viral fragments were not detectable by PCR with the subgroup III core coat protein primers and extracts of plants infected with either subgroup I or II geminivirus isolates. The demonstrated exclusive specificity of the assay for subgroup III geminiviruses offers a highly simplified PCR-based assay that permits the detection of a geographically diverse collection of WFT geminiviruses infecting cultivated crops, ornamentals, and weed hosts with minimal sample preparation. This approach is highly useful for the amplification of subgroup III geminiviral DNA templates from total nucleic acid extracts from infected plants and partially purified virion preparations.

The viral genetic basis of symptom severity and seed transmission was investigated with tobacco streak virus (TSV) pathotype I isolate Mel 40 and pathotype II isolate Mel F. These isolates differ in physicochemical properties and RNA secondary structure. Electrophoresis of RNA from the mild, infrequently seed-transmitted TSV isolate Mel F revealed many minor RNA species not detected in the severe, seed-transmitted isolate Mel 40. Inoculations with gel-purified Mel F genomic RNAs 1, 2, and 3 produced atypically severe symptoms on Chenopodium quinoa and bean (Phaseolus vulgaris). Severe symptoms were correlated with the absence of minor RNAs in the inoculum. Subisolate Mel FS arose as a rare seed transmission event of Mel F in bean. It was transmitted to approximately 30% of progeny seedlings but appeared otherwise indistinguishable from Mel F.Non-seed-transmitted Mel F encapsidated one minor RNA, designated RNA F5, which was not detected in seed-transmitted Mel FS viral RNA. Six heterologous RNA combinations from Mel F and Mel 40 were inoculated on C. quinoa by using gel-purified genomic RNAs. Only one pseudorecombinant isolate was sufficiently infectious for transfer and characterization, causing a slow-spreading infection that was not seed transmitted. Infectivity of TSV Mel 40 and Mel F appears to be dependent on homologous RNAs.

Pea streak carlavirus (PSV) incited a widespread wilting and yellowing disease of chickpea (Cicer arietinum) in commercial and experimental plantings in the Palouse region of eastern Washington and northern Idaho. Incidence of PSV usually ranged from 0.5 to 5%. Experimental host ranges of several Palouse PSV isolates were confined to the Fabaceae and one species of Amaranthaceae. Systemic necrosis developed in chickpea, lentil (Lens culinaris), pea (Pisum sativum), fenugreek (Trigonella foenum-graecum), and faba bean (Vicia faba), while alfalfa (Medicago sativa), white sweet clover (Melilotus alba), and hairy vetch (Vicia villosa subsp. villosa) were symptomless carriers of PSV

Tobacco streak ilarvirus (TSV) seed transmission was investigated by reciprocal pollinations as well as by serological and infectivity assays of flower parts from beans (Phaseolus vulgaris) systematically infected with either TSV pathotype I (isolate Mel 40) or TSV pathotype II (isolate Mel F). Heathly and TSV-infected Black Turtle Soup (BTS) bean plants were reciprocally pollinated by using anthers from either heathly plants or plants systematically infected with Mel 40 or Mel F. When anthers from plants infected with Mel 40 were used to pollinate heathly plants, a high percentage of the resulting seedlings were infected with that isolate (...)

Two pathotypes of tobacco streak virus (TSV), designated I and II, were isolated from naturally infected bean (Phaseolus vulgaris), chickpea (Cicer arietinum), adzuki bean (Vigna angularis), fenugreek (Trigonella foenum-graecum), alfalfa (Medicago sativa), and white sweet clover (Melilotus alba) at Central Ferry, WA. The tow pathotypes could be distinguished by their host reactions, serology, and by SDS-polyacrylamide gel electrophoresis. Pathotype I infected cowpea (Vigna unguiculata), but pathotype II did not (.)

Genetic diversity and incidence of seedborne bean common mosaic virus (BCMV) in Centro Internacional de Agricultura Tropical (CIAT) Phaseolus vulgaris germ plasm seed samples was compared to seed samples of the USDA accessions from which the CIAT samples were originally derived. Following approximately three seed-increase cycles at CIAT during which individual plants obviously infected with BCMV were eliminated, overall and within-sample diversity in 120 seed samples had declined by 45 and 66%, respectively. BCMV incidence also declined by 30%, from an average of 6% in 26 USDA samples to 4.2% in the CIAT samples, but tolerant and visually symptomless infections became more common in the CIAT samples (...)

Summary A new strain (R) of cowpea chlorotic mottle virus (CCMV) was isolated from plants of the resistant cowpea plant introduction 186465 which were inoculated with the type strain (T). Strain R was serologically different from five naturally occurring CCMV strains. Furthermore, it was able to replicate in and systemically invade cowpea plants which were resistant to strain T. Pseudorecombinant studies with the RNAs of strains T and R established that RNA 1 controlled systemic invasion of the resistant cowpea cultivar. Both RNA 1 and RNA 3 influenced replication in resistant cowpeas. RNA 3 also controlled the composition of the coat protein, while RNA 1 influenced symptoms in susceptible cowpeas.

Summary Recovery and specific infectivity of infectious RNA from cowpea chlorotic mottle virus of low specific infectivity (14 to 21 day infections) were greatly improved by using antioxidants during virus purification and RNA extraction, and by disrupting coat protein with pronase before phenol-SDS extraction. Total infectivity of RNA from virus of low infectivity was increased over 30 times. RNA profiles obtained using polyacrylamide gels were then similar for virus with high (4 to 7 day infections) or low specific infectivity. Low specific infectivity, therefore, seems to be caused by alteration of the coat protein or of the protein-RNA interaction in intact virus particles.